MCAT Study Guide Biology Ch. 3 – Transcription + Translation 2017-08-15T06:45:06+00:00

I.          3.1 DNA STRUCTURE

A.     General overview

1.     DNA – deoxyribonucleic acid

a)     building block is deoxyribonucloside 5’ triphosphate (dNTP)

b)     Bases:

(1)   Purines

(a)   Adenine

(b)   Guanine

(2)   Pyrimidines “cut”

(a)   Cytosine

(b)   Uracil

(c)   Thymine

(3)   A purine always pairs with a pyrimidine!

(4)   A-T, A-U; C-G (Chris Gerard is strong)

c)     Nucleosides are the sugar and base

d)     Nucleotides are nucleosides, plus phosphate groups

(1)   Polynucleotides

2.     Nucleotides are linked by phosphodiester bonds between 3’ hydroxy group of 1 deoxyribose and the 5’ phosphage group of the next deoxyribose

B.     Watson-Crick Model of DNA Structure (what about Rosalind Franklin? rude.)

1.     Cellular DNA is a right-handed double helix held together by hydrogen bonds between bases

2.     Double helix structure is antiparallel orientation

a)     5’-3’ strand lines up with 3’-5’ strand

b)     A purine always binds with a pyrimidine

(1)   A binds to T with 2 H bonds

(2)   C binds to G with 3 H bonds (therefore slightly stronger than A—T bonds)

c)     Chargoff’s rule: 

[A] = [T] and [G] = [C], and [A] + [G] = [T] + [C]

d)     Tm = Temp at which 50% DNA is melted

(1)   The more C—G bonds, the higher the Tm

3.     Chromosome – one of several large pieces of linear ds-DNA

a)     46 chromosomes, 23 pairs in humans (>109 base pairs)

b)     Single circular chromosome in bacteria (10base pairs)

c)     Circular or linear in virus

4.     DNA gyrase – enzyme in prokaryotes that uses ATP to break and twist the circular DNA chromosome, resulting in super-coiled DNA

5.     Nucleosomes – DNA wrapped around histones – like beads on a string (mostly basic so they are attracted to the acidic DNA)

 

II.          3.2 DNA’S JOB

A.     Role of DNA – to transmit the genetic information passed down from parent to offspring

B.     Genetic code

1.     Transcription – DNA to RNA

2.     Translation – RNA to protein

3.     Central Dogma – DNA → RNA → protein

a)     mRNA copies DNA

b)     mRNA travels to cytoplasm and hooks up with a ribosome

c)     The ribosome synthesizes polypeptides with the help of tRNA

4.     3 base pairs = 1 codon

a)     64 codons; 61 specify AAs, the remaining 3 are stop codons (aka nonsense codons)

b)    Many codons that have the same first 2 base pairs code for the same AA (CUU, CUA, CUG)

A.    GENETIC MUTATIONS– 3 kinds

1.     Point mutations – single base pair subsitutions

a)     Transitions (subbing one pyrimidine for another pyrimidine)

b)     Transversions (subbing a purine for a pyrimidine, or vice versa)

c)     3 subclasses:

(1)   Missense:  cause one AA to be replaces with another AA

(2)   Nonsense:  cause a stop codon to replace a regular codon

(3)   Silent:  change a codon into a new codon for the same AA

2.     Insertion mutations – the addition of one or more extra nucleotides in DNA

a)     Frameshift mutation! Very serious

3.     Deletion mutations – the removal of one or more extra nucleotides in DNA

a)     Frameshift mutation! Very serious

III.          3.3 DNA REPLICATION

B.    Rules to memorize:

1.     DNA replication is semiconservative – Messelson-Stahl

a)     DNA polymerase is the enzyme that makes a new DNA daughter strand

2.     Polymerization in 5’ → 3’ direction

3.     DNA polymerase (pol) requires a template (the parent strand)

4.     DNA polymerase requires a primer (RNA primase)

5.     Replication forks grow away from the origin in both directions:  each replication fork contains a leading and a lagging strand

a)     Leading strand replication is continuous, lagging strand replication is discontinuous, resulting in Okazaki fragments, later joined by DNA ligase

6.     Eventually all RNA primers are replaced by DNA (by DNA polymerase)

 

C.     Other facts to note

1.     Helicase unwinds and separates strands at origin of replication

2.     Topoisomerases relieves tension of the superwinding as a result of helicase by cutting the strands and reattaching them

3.     Single strand binding proteins protect the ss-DNA (called open complex), which are more unstable than ds-DNA

4.     Endonucleases cut in the middle, there are 2 types:

a)     Repair enzymes, which remove chemically damaged DNA from chain

b)     Restriction enzymes, which destroy DNA of infecting viruses

PROKARYOTIC VS EUKARYOTIC REPLICATION

Eukaryotes Prokaryotes
Chromosomes Multiple linear One circular
Origins of replication Multiple One (theta replication)
DNA polymerase 1 1.  DNA pol I (slower)

2.  DNA pol II (unknown)

3.  DNA pol III (FAST)

PROKARYOTIC VS EUKARYOTIC TRANSCRIPTION/TRANSLATION

Eukaryotes Prokaryotes
Introns/exons Yes No
RNA polymerase 1.  RNA pol I

2.  RNA pol II

3.  RNA pol III1LocationNucleusCytoplasmPrimary transcriptNot mRNA (edited first)MRNA → transcription is immediate

D.     DNA polymerase for prokaryotes:

1.     DNA poly III – Elongation of leading strand, FAST

a)     Can also proofread backwards (3’ → 5’)

2.     DNA pol II – unknown

3.     DNA pol I – same as III, but slower

a)     Can replace RNA primer using 5’ → 3’ with exonuclease activity

 

IV.          3.4 GENE EXPRESSION:  TRANSCRIPTION

A.     RNA

1.     Distinct from DNA in 3 ways:

a)     RNA is single stranded

b)     RNA contains uracil instead of thymine

c)     Pentose ring is ribose instead of 2’ dexoyribose

2.     3 Types:

a)     mRNA (messenger RNA) – molecule that carries genetic information from the nucleus to the cytoplasm where it can be translated to protein

(1)   Eukaryotic mRNA is monocistronic (each codes for one amino acid only)

(2)   Prokaryotic mRNA is polycistronic (mRNA often codes for more than 1 amino acid)

b)     rRNA (ribosomal RNA) – serve as components of the ribosome, seem to provide catalytic function (which is a little weird); there are only a few kinds

c)     tRNA (transfer RNA) – each tRNA carries one amino acid from the cytoplasm to the ribosome

B.     REPLICATION VS TRANSCRIPTION

1.     Template-driven polymerization:  daughter products (RNA, DNA) made from DNA templates and are complementary to the parent strand

a)     Driving force is removal and subsequent hydrolysis of pyrophosphate from each nucleotide added to the chain, with the existing chain acting as a nucleophile

2.     Transcription – the process synthesizing RNA using DNA as a template; is the prinicipal site of regulation of gene expression in all cells!

a)     Does not require a primer – remember, primase IS RNA

b)     RNA polymerase lacks exonuclease activity and cannot correct errors – lower fidelity rate that DNA replication

c)     Start site – analogous to the “origin” in replication

d)     Promoter – the sequence of nucleotides on a chromosome that activates RNA polymerase to begin the process of transcription (located before the start site)

C.    REFERENCE POINTS IN TRANSCRIPTION

a)     Template/non-coding/ transcribed/antisense strand – the strand which is transcribed

b)     Coding/sense strand – the strand which is complementary to the one transcribed

c)     Upstream – towards the 5’ end of the coding strand (3’ of template); negative numbers

d)     Downstream – towards the 3’ end of the coding strand (5’ of template); positive numbers

D.    PROKARYOTIC TRANSCRIPTION

1.     RNA polymerase:  large enzyme complex called the “core enzyme”; to initiate transcription, however, another subunit is required; this entire thing together is referred to as the holoenzyme

2.     3 stages:

a)     Initiation – occurs when RNA polymerase holoenzyme binds to a promotor (called Pribnow box)

(1)   RNA polymerase binds to this and forms a closed complex

(2)   RNA polymerase then unwinds some DNA (creating an open complex)

b)     Elongation – core enzyme elongates RNA chain processively, moving downstream in a transcription bubble

c)     Termination – when a signal is detected, polymerase falls off of DNA, releases RNA, and transcription bubble closes

E.     REGULATION OF PROKARYOTIC TRANSCRIPTION

1.     Primary method of regulation of gene expression!

2.     Repressible enzymes:  anabolic enzymes whose transcription is inhibited by excessive amts of product

3.     Inducible enzymes:  catabolic enzymes whose transcription can be stimulated by the abundance of substrate

4.     Operon – is a coding sequence for enzymes with upstream regulatory control

a)     Negative inducible operon – repressor protein bound to the operator which prevents transcription unless an inducer molecule is present, which binds to and removes the repressor

b)     Negative repressible operon – transcription takes place normally because the repressor protein is not active and unbound to the operator (can become active with presence of a corepressor, bind to the operator, and inhibit transcription)

c)     Positive inducible operon – activator proteins can only bind to operator if an inducer is present

d)     Positive repressible operon – activator proteins are normally bound, unless an inhibitor binds to the protein and prevents transcription

F.     COMPARING PROKARYOTIC AND EUKARYOTIC TRANSCRIPTION

1.     DIFFERENCES:

a)     Location of transcription (very important)

(1)   Eukaryotes – in nucleus

(2)   Prokaryotes – in cytoplasm (which means transcription and translation can occur simultaneously)

b)     Primary transcript and mRNA (very important)

(1)   Eukaryotes – primary transcription (of mRNA) is modified extensively before translation

(a)   mRNA often has non-coding sequences (introns )interspersed between coding portions (extrons; think ex = expressed)

(b)   Splicing – the removal of introns and joining of remaining exons

(c)   hnRNA – thought to be the primary transcript of made by RNA pol II before splicing occurs

(d)   5’ cap & 3’ poly-A tail – caps and tails placed on either end of mRNA; cap is essential for translation, and both help prevent digestion by exonucleases in cytoplasm

(2)   Prokaryotes – primary transcript is mRNA (already starts being translated before transcription is complete)

c)     RNA polymerases (sort of important) – these are responsible for making RNA

(1)   Eukaryotes – 3 kinds, 1 for each type of RNA:

(a)   RNA pol I – transcribes rRNA from DNA

(b)   RNA pol II – transcribes mRNA (think: me too [m-II])

(c)   RNA pol III – transcribes tRNA from DNA

(2)   Prokaryotes –

d)     Regulation of transcription (sort of important)

(1)   See specifics for prokaryotic regulation above, and eukaryotic, below

G.    REGULATION OF EUKARYOTIC TRANSCRIPTION

1.     TATA box – thought to be the core promoter sequence in most RNA pol II promoter sites

2.     Sequence-specific transcription factors (SSTFs) – proteins that bind to certain sequences in DNA and  either increase or decrease transcription

3.     Enhancer – a sequence that may be located very far away from the promotor (either upstream or downstream) and still regulate transcription (SSTFs may bind to these too)

IV.          3.5:  GENE EXPRESSION:  TRANSLATION

The synthesis of polypeptides according to the AA sequence dictated by the mRNA; mRNA attaches to a ribosome, then a tRNA delivers the appropriate AA, then another brings the next; the ribosome binds the 2 AAs together

A.     Transfer RNA (tRNA)

1.     Produced by RNA pol III

2.     Cloverleaf structure

3.     Anticodon – the end of the tRNA which is complementary to the codon in tRNA translation (complementary to the mRNA it is attaching to)

4.     AA receptor site – the opposite end of tRNA (from anticodon) where the AA is attached to (always CCA at 3’ end)

5.     Peptide bond formation:

a)     Unfavorable thermodynamics (ΔG > 0)

b)     Slow kinetics (high activation energy)

B.     Aminoacyl-tRNA Synthetase

1.     This is an enzyme that binds the aminoacyl-AMP to the tRNA

2.     Must recognize both the tRNA and AA based on the 3D structure

3.     There is at least 1 Aminoacyl-tRNA synthetase for every AA

4.     Must be highly specific

C.    AA activation (through coupling – 2 ATPs):

1.     Step 1 – AA attaches to ATP

2.     Step 2 – phosphate group leaves and is hydrolized to 2 orthophosphates (very favorable,  ΔG << 0)

3.     Step 3 – tRNA loading is very unfavorable, but is driven forward by the breaking of the aminoacyl-AMP bond, which is high energy

4.     Step 4 – eventually, the AA-tRNA bond will be broken and this will power the peptide bond formation

D.    The Ribosome

1.     Composed of many polypeptides and rRNA chains in quarternary structure

2.     Eukaryotes – 80S ribosome (subunits are 60S and 40S)

3.     Prokaryotes – 70S ribosome (subunits are 50S and 30S)

4.     Complete ribosome (both subunits) have 3 binding sites:

a)     A site (aminoacyl-tRNA site) – where tRNA binds to deliver the AA

b)     P site (peptidyl-tRNA site) – where the growing polypeptide chain, still attached to tRNA is located during translation

c)     E site (exit tRNA site) – where the now-empty tRNA site prior to its release from the ribosome

E.     Prokaryotic Translation

1.     Occurs in same compartment and at the same time as transcription

2.     Multiple ribosomes attach in different locations (remember, mRNA can code for different proteins)

3.     Shine-Dalgarno sequence – ribosome binding site located about 10 units upstream from start site

4.     3 steps of Translation:

a)     Initiation

(1)   1st:  requires the formation of the 70S complex from the 30S and 50S (1 GTP)

(2)   fMet-tRNA – the first amoniacyl-tRNA that sites at the P site with the start codon (must be preceded by Shine-Dalgarno sequence)

b)     Elongation – 3 step cycle

(1)   1 – Second aminoacyl-tRNA enters the A site and H-bonds with 2nd codon (1 GTP)

(2)   2 – peptidyl transferase activity catalyzes formation of peptide bond between fMet and 2nd AA; tRNA dissociates from the ribosome and the new dipeptide is attached to 2nd AA

(3)   3 – translocation:  tRNA #1 (now empty) moves to E site, then tRNA #2 moves to P site, and new codon can land in A site (1 GTP)

c)     Termination

(1)   Occurs when stop codon appears in A site

(2)   Instead of tRNA, a release factor now enters the A site, causing peptidyl transferase to hydrolyze the bond between the last tRNA and the completed polypeptide

(3)   Ribosome subunits separate and release mRNA and peptide

F.     Eukaryotic Translation

1.     Differences from prokaryotes:

a)      Ribosome is larger

b)     mRNA processed before translation occurs (spliced, tail and cap added), then transported

c)     N-terminal AA is different (MET instead of f-Met)

d)     Other sequences besides Shine-Dalgarno sequence used to initiate (like Kozak)

e)     Order in which initiaion complex is formed is different:

(1)   1st, tRNA binds to smal subunit, then mRNA binds to small subunit, then large subunit binds

MCAT Study Guide Biology - Kim Matsumoto


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